Mechanical nanosurgery of chemoresistant glioblastoma using magnetically controlled carbon nanotubes

Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Despite multimodal treatment including surgery, radiotherapy, and chemotherapy, median patient survival has remained at ~15 months for decades. This situation demands an outside-the-box treatment approach. Using magnetic carbon nanotubes (mCNTs) and precision magnetic field control, we report a mechanical approach to treat chemoresistant GBM. We show that GBM cells internalize mCNTs, the mobilization of which by rotating magnetic field results in cell death. Spatiotemporally controlled mobilization of intratumorally delivered mCNTs suppresses GBM growth in vivo. Functionalization of mCNTs with anti-CD44 antibody, which recognizes GBM cell surface–enriched antigen CD44, increases mCNT recognition of cancer cells, prolongs mCNT enrichment within the tumor, and enhances therapeutic efficacy. Using mouse models of GBM with upfront or therapy-induced resistance to temozolomide, we show that mCNT treatment is effective in treating chemoresistant GBM. Together, we establish mCNT-based mechanical nanosurgery as a treatment option for GBM.


I. Comparison of two types of mCNTs
For mCNTs, magnetic materials such as iron particles can be either on the surface or inside the nanotubes. The toxicity effects and treatment effects of these two types of mCNTs were compared. Three concentrations of mCNTs (n = 3 independent repeated experiments for each of the three concentrations 0.01 mg/mL, 0.025 mg/mL, 0.05 mg/mL) were used in experiments and treated as a block condition in statistical analysis. The results showed a significant lower percentage of cell death ratio using mCNT2 (iron inside) compared with mCNT1 (iron on surface) (9.93 ± 3.21% vs. 22.14 ± 14.27%, error bar: standard deviation, P = 0.0235) ( Figure   S1b), demonstrating the lower toxicity to the cells from mCNT2.
The treatment effect under the same magnetic field treatment was quantified and compared between two types of mCNTs. The treatment effect was quantified as the subtraction between cell death ratio after magnetic treatment and cell death ratio without magnetic treatment (cell death solely caused by toxicity from mCNTs).
Similar to the quantification of toxicity, three concentrations were used to treat the cells (n = 3 independent repeated experiments for each of the three concentrations 0.01 mg/mL, 0.025 mg/mL, 0.05 mg/mL) and used as blocking conditions in statistical analysis. The results showed no significant difference in percentage of cell death ratio caused by magnetic treatment using mCNT2 compared with mCNT1 (18.11 ± 5.75% vs. 19.98 ± 3.02%, error bar: standard deviation, P = 0.4016) (Figure S1c). mCNT2 with iron inside was chosen for all subsequent experiments.

II. Magnetic field parameters
When subjected to a magnetic field, mCNTs align with direction of the magnetic field (i.e., the direction of magnetic flux density B), and rotate with the rotating magnetic field. In magnetic field control, a mCNT align itself with direction of the magnetic field strength through torque, which depends on magnetic moment of the mCNT and magnitude of the magnetic field strength.
where T is torque, m is magnetic moment on the mCNT which depends on magnetic property of the mCNT (e.g., concentration of iron) and mass, B is field flux density, and θ is angle between mCNT and the magnetic field. The rotation aligns m with B, minimizing θ until it reaches zero. When mCNTs are in the rotating magnetic field, the magnetic torque, determined by the magnitude of magnetic field flux density, rotates mCNT to follow rotation of the magnetic field. To estimate the rotating motion of mCNT, the mechanical work is where ω is angular velocity, I is moment of the inertia around the axis of rotation depending on the mass and geometry of mCNT (Figure S1d), W rotational is mechanical work applied through the rotational motion. Based on the rotational energy equation, the angular frequency of the rotation of mCNT would determine the rotational energy delivered to cells over a 30-minute treatment duration.
The rotational motion of mCNTs applies mechanical stimulation to intracellular structures (e.g., intracellular membranes and cytoskeleton). Based on equations (1) and (2) The magnetic field used in treatment was in the range of 20 to 40 mT, with a magnetic gradient smaller than 2 T/m. For an iron particle clump with a diameter 16.25 ± 3.24 nm, the magnetic gradient force for pulling iron particle clump towards outside of carbon nanotube is smaller than 2.1 × 10 -15 N. TEM with EDX before and after magnetic field actuation showed that there was no significant difference in iron content within carbon nanotubes, indicating that the applied magnetic field actuation did not cause iron content loss.
The size and iron concentration can affect the motility performance of mCNTs. Because of the larger moment of inertia and larger magnetic moment, a longer size and higher iron concentration of mCNTs can potentially result in larger mechanical work delivered to the cell under the same magnetic field. However, a longer size and higher iron concentration could also change the frequency response of the mCNTs and/or lower the amount of mCNTs internalized by the cell. Future work to optimize mCNT length and iron concentration may further enhance treatment efficacy.

III. Magnetic field generation for in vivo treatment
Mechanical work exerted by mCNTs may cause cell death in the healthy region surrounding the tumor region.
Hence, our treatment field was designed to maintain sufficient rotational energy within tumor while minimizing rotational energy outside the tumor region. The techniques include coordinate transformation from stereotaxic coordinates to Cartesian coordinates (through top-down and side view of the bioluminescence images), and magnetic field modeling for confining the region with a rotating magnetic field. In the magnetic field treatment system, the position of each magnetic pole was captured by camera mounted on the system with coordinates within the Cartesian coordinate system; when a mouse was placed within the workspace between each of the magnetic poles, the Cartesian coordinate of mouse's eyes and nose tip were also captured by the camera (Figure 2f). The location of tumor center (i.e., Cartesian coordinate) was calculated through its relative position to mouse's eyes and nose top based on top-down and side view of the bioluminescent image (Figure 2g). Based on the location of tumor center and tumor size, the magnetic field model was used to generate a rotating magnetic field within tumor while minimizing field strength in brain region surrounding the tumor.
In the magnetic field control strategy ( Figure S7), a rotating magnetic field with a field strength maintained at 20 mT was applied to tumor region to induce tumor cell death, while the magnetic field strength was lower outside the tumor region. In the first quarter of a control cycle (0-1/4T), a pair of adjacent coils function as dominant coils for generating the rotating magnetic field. Meanwhile, the other pair of coils act as auxiliary coils to attenuate the magnetic field outside the target region. In the subsequent quarter of the same cycle (1/4T-1/2T), dominant coils and auxiliary coils are shifted clockwise by one coil. In this quarter, the magnetic field distribution changes, and the non-tumor region treated in the previous quarter, is now located in a region with low magnetic field strength. Dominant coils shift four times to complete a full control cycle T. Throughout a control cycle, the magnetic field in target region is always sufficiently large, maintaining strong treatment effects. However, outside the target region, the non-tumor region is periodically (a quarter of a cycle) subjected to high and low magnetic field strength ( Figure S7b). As such, only the specified tumor region is subjected to strong rotating field strength throughout the treatment cycle, whereas the non-tumor region is only subjected to a quarter of the cycle. A spherical magnetic insulation was imposed to enclose the model, which set the tangential components of the magnetic potential to zero at the boundary. In the model, each coil was wired with Gauge 16 copper wires with a conductivity of 6×10 7 S/m, and the number of turns was set as 900 as in our experimental system. The material of iron core was assigned to be 4140 steel with a relative permeability of 700. A series of sinusoidal electrical current sequences, as determined by our magnetic field control strategy, were supplied to coils to generate rotational magnetic field. The maximum amplitude of electrical current was set at 5 A based on hardware limitation of our custom-designed current amplifier. The current waveform supplied to each coil was shown in Figure S7a. Three mesh sizes were tested. "Fine", "Finer" and "Extra Fine" (defined by COMSOL) meshes with a mesh size of 2.4 mm, 0.96 mm, and 0.36 mm, respectively, were used for comparison. The maximum differences between three computational meshes were less than 1% for magnetic field strength within the workspace.

IV. Finite element simulation for estimating rotational energy delivered
In simulation, in a tumor region of 2.8 mm in radius and mCNTs of 0.48 µm in length and 52 wt% in iron percentage, the designed target magnetic field delivers a total of 2.5 × 10 -16 J rotational energy to tumor during one-hour treatment, compared with 2.55 × 10 -16 J under a uniform rotating magnetic field of 20 mT field strength. In the tissue surrounding the tumor, the rotational energy delivered was limited to 0.16 × 10 -16 J ( Figure S7).

V. Mechanical environment and treatment efficacy
The in vitro and in vivo mechanical environments are different. For instance, plastic petri dish has a stiffness about 2 GPa, and brain tumor tissue has a stiffness ranging from 500 to 10,000 Pa in vivo (69). To study whether microenvironmental stiffness difference can cause variations in treatment efficacy, we cultured GBM cells on substrates with different mechanical stiffness (200, 500, 1,000 Pa) and treated the cells with mCNT and magnetic field. The results (Figure S18) showed significant increase in treatment efficacy with the decrease of substrate stiffness.                mCNTs localize within the extracellular space, on cell membrane, and within the cell. We propose that mechanical torque from magnetic actuation can be transmitted from extracellular space to inside of the cell, generated directly on cell membrane, and generated within the cell to mechanically damage intracellular organelles, increase DNA damage, and induce cell apoptosis.